The calcium-binding photoprotein is present as a complex of an apoprotein and the peroxide of coelenterazine as a light-emitting substrate. The calcium-binding photoprotein emits light when the protein binds calcium ions. Among the calcium-binding photoproteins, aequorin, obelin, clytin, mitrocomin, mineopsin. bervoin, etc. are well-characterized.
Aequorin is a representative protein in the calcium-binding photoproteins, and its protein structure and the luminescence mechanism, etc. have been reported in detail (cf, e.g., Inouye et al (1985) Proc. Natl. Acad. Sci. USA 82, 3154-3158; Head et al. (2000) Nature 405, 372-376; etc.). The homology of the primary structures among the calcium-binding photoproteins is very high, and the luminescence mechanism and protein structure are found to be essentially the same, based on the analysis of the crystal structures of aequorin (PDB: 1EJ3), obelin (PDB: 1EL4, 1JF0, 1QV0) and clytin (PDB: 3KPX). Also, aequorin has an extremely high sensitivity to calcium ions and is used for the detection and quantification of a trace amount of calcium ions, assay for intracellular calcium ion changes, etc.
On the other hand, it is known that the calcium-binding domains in photoproteins have a unique structure, generally termed EF hand structure, which is present in many calcium-binding proteins. That is, the calcium-binding domains in photoproteins have a loop structure which is consisted of alpha helix-loop-alpha helix, and 12 amino acid sequence in the loop structure contains the following consensus sequence: O—O—OGly-Ile(Leu)-O—O (O: oxygen atom-containing amino acid, -: any amino acid). Until now, many aequorin mutants have been constructed by site-directed mutagenesis and random mutagenesis and luminescence activities or luminescence patterns of the aequorin mutants have been analyzed (Tsuji et al (1986) Proc. Natl. Acad. Sci. 83, 8107-8111; Kurose et al. (1989) Proc. Natl. Acad. Sci. 86, 80-84; Kendall et al. (1992) Biochem. Biophys. Res. Co mMun. 187, 1091-1097; Ohmiya et al (1992) FEBS Lett. 301, 197-201; Ohmiya et al (1993) FEBS Lett. 320, 267-270; Tsuzuki et al. (2005) J. Biol. Chem. 280, 34324-34331; Tricoire et al. (2006) Proc. Natl. Acad. Sci. 103, 9500-9505; etc.).
Among these photoproteins, it has been reported that some photoproteins show a rapid decay pattern of luminescence and some photoproteins show a slow decay pattern of luminescence by adding calcium. All of the aequorin mutants showing a slow decay pattern of luminescence that have been reported so far are obtained by substitution of amino acids containing oxygen atoms in the loop region of the EF hand structure (Kendall et al. (1992) Biochem. Biophys. Res. Co mMun. 187, 1091-1097; Tsuzuki et al. (2005) J. Biol. Chem. 280, 34324-34331; Tricoire et al. (2006) Proc. Natl. Acad. Sci. 103, 9500-9505; etc.).
Aequorin has been widely used in the dynamic analysis of intracellular calcium ions for screening the target compound for G protein-coupled receptors in drug development. In recent years, much attention has been focused on a method for screening a target compound using semisynthetic aequorins showing a slow decay pattern of luminescence, namely, with less sensitivity to calcium, and a screening method using analogues of coelenterazine as a light-emitting substrate has been established (Inouye et al. (2010) Anal. Biochem. 407, 247-252; etc.).
On the other hand, there is also desired an aequorin with low calcium sensitivity in which a decay of the luminescence can be slowed down even when native coelenterazine or h-coelenterazine is used as a light-emitting substrate.